Ebola virus is a Category A virus and an emerging human pathogen that causes devastating outbreaks of hemorrhagic fever. Assembly and budding are important late events in the replication cycle of Ebola virus. While much has been learned regarding these dynamic and multifaceted stages of virus replication, the molecular mechanisms of assembly and budding remain unclear. This proposal focuses on the VP40 matrix protein of Ebola virus. Ebola VP40 protein contains late budding domains (L-domain) that play a key role in virus egress by mediating interactions with host proteins, such as tsg101. We hypothesize that host innate immune responses inhibit Ebola virus budding by targeting VP40 L-domain activity. Preliminary data implicate interferon stimulated gene 15 (ISG15) in inhibiting Ebola VP40 VLP budding. To test our hypothesis, we propose 3 Specific Aims: 1) To determine whether expression of ISG15 inhibits release of Ebola VP40 VLPs in an L-domain dependent manner, 2) To determine whether expression of ISG15 inhibits release of Ebola virus or VSV recombinants expressing Ebola virus L-domains, and 3) To determine whether expression of ISG15 inhibits Ebola VP40/host tsg101 interactions. Results from these studies should provide fundamental knowledge of how specific components of the innate immune response may inhibit Ebola virus budding, and provide the foundation for future studies to elucidate further the mechanisms and biological significance of these innate immune responses to Ebola virus infection.